Longer exaggerated male genitalia confer defensive sperm-competitive benefits in an earwig

As evidence mounts that male genitalia can affect relative fertilisation success, the role that sexual selection has played in the rapid and divergent evolution of genitalia is becoming increasingly recognized. Unfortunately, the limited functional understanding of these complex structures and their interactions with the female reproductive tract often limit interpretation regarding their evolution. Here, we address this issue using the earwig Euborellia brunneri, where both the male intromittent organ and the female spermatheca are highly exaggerated in length yet structurally simple. In a double mating design, we use the sterile male technique to study how sperm precedence patterns are affected by male genital length, male age, and the size of the male sperm storage organ, the seminal vesicle. Relative fertilisation success exhibited considerable variation around modest last-male paternity. Only an interaction between first and second male genital length affected paternity, where males gained reduced paternity when preceded by rivals with longer genitalia. Longer genitalia confer defensive benefits in sperm competition by apparently depositing ejaculate deeper in the tubular spermatheca, safe from removal by rivals. Paternity similarly depended on an interaction between the ages of both males, likely mediated by sperm traits as testes size decreased with age. Seminal vesicle size showed positive allometry but did not affect paternity; instead, greater seminal vesicle size in last males expedited oviposition. The exaggerated yet relatively simple genitalia of E. brunneri facilitate an unusually clear example of post-copulatory selection on phenotypic variation in multiple reproductive traits.     

Postmating sexual selection: the effects of male body size and recovery period on paternity and egg production rate in a water strider

The role of male body size in postmating sexual selection wasexplored in a semiaquatic insect, the water strider Gerris lateralis.To separate effects of male size per se from those due to numericsperm competition, male recovery period (shown here to be proportionalto ejaculate size) was manipulated independently of body sizein a factorial experiment where virgin females were mated firstwith sterile males and then with focal males. Both relativemale fertilization success and female reproductive rate were measured.The number of sperm transferred increased with male recoveryperiod, an effect that was mediated by longer copulation duration,but there were no effects of body size on ejaculate size. Neithermale size nor recovery period had any significant direct effectson male fertilization success. However, copulation durationinfluenced relative fertilization success, suggesting that malesable to transfer more sperm also achieved higher fertilizationsuccess. Females exercised cryptic female choice by modulatingtheir reproductive rate in a manner favoring large males andmales that were successful in terms of achieving high relativefertilization success. Thus, successful males gained a twofoldadvantage in postmating sexual selection. This study has important implicationsfor previous estimates of sexual selection in this group of insectsbecause pre- and postmating sexual selection will be antagonisticdue to limitations in male sperm production: males mating frequently(high mating success) will on average transfer fewer sperm ineach mating and will hence tend to fertilize fewer eggs permating (low fertilization success).     

Influence of body and genital morphology on relative male fertilization success in oriental beetle

Although the frequently large variance in relative male fertilizationsuccess when females are mated by more than 1 male has beenappreciated for some time, the factors that influence relativepaternity are still poorly understood. Recently, experimentalevidence that morphology of male genitalia influences fertilizationsuccess has been documented in 2 water striders, a dung beetle,and a leaf beetle. We explored the role of male genital morphologyin postcopulatory sexual selection in the oriental beetle. Wemated females to 2 males in succession and assessed relativepaternity by the sterile male technique. Morphology of the malegenitalia was found to strongly influence relative paternitybut only for the first male to mate. Male body size influencedrelative fertilization success as well, but again, only forthe first male; surprisingly, smaller males achieved higherpaternity when mating first. We also found suggestive evidencethat copula duration of both the first and second male to mateinfluenced paternity. Other factors, including female size anddegree of asymmetry of hind tibiae length of males had no effecton relative fertilization success. Our results for the orientalbeetle are novel among sperm precedence studies for 2 reasons:1) traits of the first male appear to be more important in influencingpaternity than those of the second, and 2) smaller, not larger,males achieved greater relative paternity. Our results alsocontribute to the growing body of empirical evidence in supportof the hypothesis that male genitalia evolve by postcopulatorysexual selection.     

Sexual selection in flour beetles: the relationship between sperm precedence and male olfactory attractiveness

Sperm precedence, defined as nonrandom differential fertilizationsuccess among mating males, is an important postmating componentof sexual selection. This study examined the relationship betweenpremating and postmating components of sexual selection in malesof the flour beetle (Tribolium castanewn). Male olfactory attractivenessto females was positively correlated with a male's subsequentfertilization success: more attractive males achieved highersecond-male sperm precedence when allowed to mate with previouslyinseminated females. Attractive males may achieve compoundedgains in their reproductive success through enhanced matingopportunities as well as through greater fertilization success.Thus, the relationship between these reproductive fitness componentsmay augment differences in reproductive success among males.Female fecundity, estimated as the number of adult progeny produced,increased significantly with multiple malings. This result supportsincreased female reproductive success as a direct benefit ofmultiple mating in T. caslaneum and suggests that progeny productionis partially limited by sperm availability. Total progeny productionby doubly mated females remained constant at all levels of second-malesperm precedence. However, higher sperm precedence was associatedwith a decline in firstmale progeny and a concomitant increasein second-male progeny. This pattern of progeny production suggeststhat more attractive males may achieve higher fertilizationsuccess through a combination of displacement of previouslystored sperm, transfer of greater sperm quantities, or females'preferential use of sperm of attractive males for fertilizations.     

The intensity of sexual selection in relation to male sexual behaviour,female choice,and sperm precedence

In this paper we define sexual selection on males as the variance in numbers of mates per male and show how the intensity of this selection is affected by male sexual behaviour, female choice, sex ratio, and modes of sperm precedence. This definition coincides with Darwin's conception of sexual selection but differs from some post-Darwinian views. For systems of single-male paternity, we show that the intensity of total selection on male reproductive success equals the intensity of natural selection on female fertility, times the sex ratio, plus the intensity of sexual selection on males. The absolute intensity of sexual selection is unaffected by the system of sperm precedence. The application of the results to field studies is discussed.     

Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

Size-dependent mating strategies and the risk of cannibalism

The role of sexual selection in determining the nature and direction of sexual size dimorphism may depend upon the timing of sexual selection, and this may also influence the variation in male size. For example, selection through sperm competition favours smaller males in the highly sexually size dimorphic orb-weaving spider Nephila edulis , whereas larger males are better able to exclude their smaller rivals from the central hub of the web where mating takes place. We investigate experimentally the role of body size and hub tenure in determining male fertilization success when males of different sizes compete for a single female over a 24-h period that includes a period of darkness. Our results confirm that small and large males obtain similar paternity share but that, in contrast with previous studies, hub tenure does not translate into greater paternity share. Unexpectedly, smaller males are at greater risk of postmating sexual cannibalism than larger males, suggesting that natural selection through sexual cannibalism may place a lower limit on male size.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 355–363.     

Female choice, male interference, and sperm precedence in the red-spotted newt

Darwin first identified female choice and male—male competitionas forms of sexual selection resulting in the evolution of conspicuoussexual dimorphism, but it has proven challenging to separatetheir effects. Their effects on sexual selection become evenmore complicated when sperm competition occurs because spermprecedence may be either a form of cryptic female choice ora form of male—male competition. We examined the effectsof tail height on male—male competition and female choiceusing the sexually dimorphic red-spotted newt (Notophthalmusviridescens viridescens). Experiment 1 examined whether maletail height influenced male mating success. Males with deeptails were more successful at mating with females than thosewith shallow tails. Successful, deep-tailed males also were bigger(snout-vent length; SVL) than unsuccessful, shallow-tailed males,but they did not vary in tail length or body condition. Of these,only tail height and tail length are sexually dimorphic traits.Experiment 2 tested the hypothesis that the differential successof males with deeper tails was due to female choice by examiningboth simultaneous female preference for association and sequentialfemale choice. We found no evidence of female choice. When maleswere not competing to mate with females, tail height did notinfluence male mating success. Successful males did not havedifferent SVL and tail lengths than unsuccessful males. Thus,tail height in male red-spotted newts appears to be an intrasexuallyselected secondary sexual characteristic. Experiment 3 usedpaternity exclusion analyses based on molecular genetic markersto examine the effect of sperm precedence on sperm competitionin doubly-mated females. Sperm precedence likely does not havea pervasive and consistent effect on fertilization success becausewe found evidence of first, last, and mixed sperm usage.     

Influence of developmental environment on male- and female-mediated sperm precedence in Drosophila melanogaster

Length of the sperm flagellum and of the female's primary sperm-storage organ, the seminal receptacle (SR), exhibit a pattern of rapid correlated evolution in Drosophila and other lineages. Experimental evolution studies with Drosophila melanogaster indicate that these traits have coevolved through sexual selection, with length of the SR representing the proximal basis of female sire discrimination, biasing paternity according to sperm length. Here, we examine the impact of experimentally varying the developmental environment, including larval density and larval and adult nutrition, on sperm length, SR length and on the pattern of sperm precedence. Expression of SR length was far more sensitive to variation among developmental environments than was sperm length. Nevertheless, there was striking co-variation in sperm and SR length. The developmental environment of both females and second males, but not first males, significantly contributed to variation in male competitive fertilization success.     

Sperm competition, alternative mating tactics and context-dependent fertilization success in the burying beetle, Nicrophorus vespilloides

Fertilization success in sperm competition is often determined by laboratory estimates of the proportion of offspring sired by the first (P1) or second (P2) male that mates. However, inferences from such data about how sexual selection acts on male traits in nature may be misleading if fertilization success depends on the biological context in which it is measured. We used the sterile male technique to examine the paternity of the same male in two mating contexts in the burying beetle, Nicrophorus vespilloides, a species where males have alternative mating strategies based on the presence or absence of resources. We found no congruence in the paternity achieved by a given male when mating under different social conditions. P2 estimates were extremely variable under both conditions. Body size was unrelated to success in sperm competition away from a carcass but, most probably through pre-copulatory male-male competition, influenced fertilization success on a carcass. The contribution of sperm competition is therefore dependent on the conditions under which it is measured. We discuss our findings in relation to sperm competition theory and highlight the need to consider biological context in order to link copulation and fertilization success for competing males.     

Genital morphology and fertilization success in the dung beetle Onthophagus taurus: an example of sexually selected male genitalia

In animals with internal fertilization and promiscuous mating, male genitalia show rapid and divergent evolution. Three hypotheses have been suggested to explain the evolutionary processes responsible for genital evolution: the lock-and-key hypothesis, the pleiotropy hypothesis and the sexual-selection hypothesis. Here, we determine whether variation in male genital morphology influences fertilization success in the dung beetle Onthophagus taurus, as predicted by the sexual-selection hypothesis. Variation in four out of five genital sclerites of the endophallus influenced a male's fertilization success, supporting the general hypothesis that male genitalia can evolve under sexual selection. Furthermore, different genital sclerites were found to enhance first versus second male paternity, indicating that different sclerites serve offensive and defensive roles. Genital-trait variability was comparable to that in other species but was less variable than a non-genital sexually selected trait (head horns). We suggest that directional selection for genital elaboration may be countered by natural selection, which should favour genitalia of a size and shape necessary for efficient coupling and sperm transfer.     

Paternity analyses in wild‐caught and laboratory‐reared Caribbean cricket females reveal the influence of mating environment on post‐copulatory sexual selection

Polyandry is ubiquitous in insects and provides the conditions necessary for male‐ and female‐driven forms of post‐copulatory sexual selection to arise. Populations of Amphiacusta sanctaecrucis exhibit significant divergence in portions of the male genitalia that are inserted directly into the female reproductive tract, suggesting that males may exercise some post‐copulatory control over fertilization success. We examine the potential for male–male and male–female post‐copulatory interactions to influence paternity in wild‐caught females of A. sanctaecrucis and contrast our findings with those obtained from females reared in a high‐density laboratory environment. We find that female A. sanctaecrucis exercise control by mating multiple times (females mount males), but that male–male post‐copulatory interactions may influence paternity success. Moreover, post‐copulatory interactions that affect reproductive success of males are not independent of mating environment: clutches of wild‐caught females exhibit higher sire diversity and lower paternity skew than clutches of laboratory‐reared females. There was no strong evidence for last male precedence in either case. Most attempts at disentangling the contributions of male–male and male–female interactions towards post‐copulatory sexual selection have been undertaken in a laboratory setting and may not capture the full context in which they take place – such as the relationship between premating and post‐mating interactions. Our results reinforce the importance of designing studies that can capture the multifaceted nature of sexual selection for elucidating the role of post‐copulatory sexual selection in driving the evolution of male and female reproductive traits, especially when different components (e.g. precopulatory and post‐copulatory interactions) do not exert independent effects on reproductive outcomes.     

Sperm competition and the evolution of precopulatory weapons: Testis size and amplexus position,but not arm strength,affect fertilization success in a chorusing frog

Trade‐offs between pre‐ and postcopulatory traits influence their evolution, and male expenditure on such traits is predicted to depend on the number of competitors, the benefits from investing in weapons, and the risk and intensity of sperm competition. Males of the chorusing frog Crinia georgiana use their arms as weapons in contest competition. Previously, we showed that increased numbers of rivals elevated the risk and intensity of sperm competition due to multimale amplexus, and caused a reversal in the direction of precopulatory selection on arm girth. Here, we focused on the factors affecting postcopulatory fertilization success during group spawning, using paternity data from natural choruses. Competitive fertilization success depended on the time spent amplexed and amplexus position. Relative testes size but not arm girth, contributed to fertilization success, but the effect of testes size depended on amplexus position. Our findings offer within species empirical support for recent sperm competition models that incorporate precopulatory male–male competition, and show why an understanding of the evolution of animal weapons requires a consideration of both pre‐ and postcopulatory episodes of sexual selection.     

Resolving mechanisms of short-term competitive fertilization success in the red flour beetle

Postcopulatory sexual selection occurs when sperm from multiple males occupy a female’s reproductive tract at the same time and is expected to generate strong selection pressures on traits related to competitive fertilization success. However, knowledge of competitive fertilization success mechanisms and characters targeted by resulting selection is limited, partially due to the difficulty of discriminating among sperm from different males within the female reproductive tract. Here, we resolved mechanisms of competitive fertilization success in the promiscuous flour beetle Tribolium castaneum. Through creation of transgenic lines with fluorescent-tagged sperm heads, we followed the fate of focal male sperm in female reproductive tracts while tracking paternity across numerous rematings. Our results indicate that a given male’s sperm persist and fertilize eggs through at least seven rematings. Additionally, the proportion of a male’s sperm in the bursa (the site of spermatophore deposition), which is influenced by both timing of female’s ejecting excess sperm and male size, significantly predicted paternity share in the 24 h following a mating. Contrary to expectation, proportional representation of sperm within the female’s specialized sperm-storage organ did not significantly predict paternity, though spermathecal sperm may play a role in fertilization when females do not have access to mates for longer time periods. We address the adaptive significance of the identified reproductive mechanisms in the context of T. castaneum’s unique mating system and ecology.     

Male and female effects on sperm precedence in the giant sperm species <Emphasis Type="Italic">Drosophila bifurca</Emphasis>

Sperm competition is expected to be a driving force in sexual selection. In internally fertilized organisms, it occurs when ejaculates from more than one male are present simultaneously within the female’s reproductive tract. It has been suggested that greater sperm size may improve the competitive ability of sperm, but studies provide contradictory results depending on the species. More recently, the role of females in the evolution of sperm morphology has been pointed out. We investigate here the male and female effects that influence sperm precedence in the giant sperm species, Drosophila bifurca Patterson & Wheeler. Females were mated with two successive males, and the paternity outcomes for both males were analyzed after determining sperm transfer and storage. We found very high values of last male sperm precedence, suggesting a strong interaction between rival sperm. However, the data also indicate high frequencies of removal of the sperm of the first male from the female reproductive tract prior to any interaction with the second male. This implies that successful paternity depends mainly on successful sperm storage. Knowing what happens to the sperm within females appears to be a prerequisite for disentangling post-copulatory sexual interactions between males and females.     

Pre‐ and postcopulatory sexual selection act in concert to determine male reproductive success in Tribolium castaneum

To capture how sexual selection shapes male reproductive success across different stages of reproduction in Tribolium castaneum (Coleoptera, Tenebrionidae), we combined sequential sperm defence (P1) and sperm offence (P2) trials with additional trials where both males were added simultaneously to the female. We found a positive correlation between the relative paternity share in simultaneous male–male competition trials and the P2 trial. This suggests that males preferred by females as sires achieve superior fertilization success during sperm competition in the second male position. In simultaneous male–male competition trials, where pre‐, peri‐ and postcopulatory sexual selection were all allowed to act, the relative paternity share of preferred males was more than 20% higher than in P2 sperm competition trials where precopulatory female choice was disabled. Additional behavioural observations revealed that mating with more attractive males resulted significantly more frequently in offspring production than mating with less attractive males. Thus, by comparing male fertilization success in trials where precopulatory choice was turned off with more inclusive estimates of fertilization success where pre‐ and pericopulatory choice could occur, we show that female mate choice may effectively inhibit sperm competition. Female mate choice and sperm competition (P2) are positively correlated, which is consistent with directional sexual selection in this species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 67–75.     

Pre‐ and postcopulatory sexual selection favor aggressive,young males in polyandrous groups of red junglefowl

A challenge in evolutionary biology is to understand the operation of sexual selection on males in polyandrous groups, where sexual selection occurs before and after mating. Here, we combine fine‐grained behavioral information (>41,000 interactions) with molecular parentage data to study sexual selection in replicated, age‐structured groups of polyandrous red junglefowl, Gallus gallus. Male reproductive success was determined by the number of females mated (precopulatory sexual selection) and his paternity share, which was driven by the polyandry of his female partners (postcopulatory sexual selection). Pre‐ and postcopulatory components of male reproductive success covaried positively; males with high mating success also had high paternity share. Two male phenotypes affected male pre‐ and postcopulatory performance: average aggressiveness toward rival males and age. Aggressive males mated with more females and more often with individual females, resulting in higher sexual exclusivity. Similarly, younger males mated with more females and more often with individual females, suffering less intense sperm competition than older males. Older males had a lower paternity share even allowing for their limited sexual exclusivity, indicating they may produce less competitive ejaculates. These results show that—in these populations—postcopulatory sexual selection reinforces precopulatory sexual selection, consistently promoting younger and more aggressive males.     

Sperm mobility: mechanisms of fertilizing efficiency, genetic variation and phenotypic relationship with male status in the domestic fowl, Gallus gallus domesticus

When females are sexually promiscuous, sexual selection continues after insemination through sperm competition and cryptic female choice, and male traits conveying an advantage in competitive fertilization are selected for. Although individual male and ejaculate traits are known to influence paternity in a competitive scenario, multiple mechanisms co-occur and interact to determine paternity. The way in which different traits interact with each other and the mechanisms through which their heritability is maintained despite selection remain unresolved. In the promiscuous fowl, paternity is determined by the number of sperm inseminated into a female, which is mediated by male social dominance, and by the quality of the sperm inseminated, measured as sperm mobility. Here we show that: (i) the number of sperm inseminated determines how many sperm reach the female sperm-storage sites, and that sperm mobility mediates the fertilizing efficiency of inseminated sperm, mainly by determining the rate at which sperm are released from the female storage sites, (ii) like social status, sperm mobility is heritable, and (iii) subdominant males are significantly more likely to have higher sperm mobility than dominant males. This study indicates that although the functions of social status and sperm mobility are highly interdependent, the lack of phenotypic integration of these traits may maintain the variability of male fitness and heritability of fertilizing efficiency.     

RAPID DIVERSIFICATION OF SPERM PRECEDENCE TRAITS AND PROCESSES AMONG THREE SIBLING DROSOPHILA SPECIES

Postcopulatory sexual selection is credited with driving rapid evolutionary diversification of reproductive traits and the formation of reproductive isolating barriers between species. This judgment, however, has largely been inferred rather than demonstrated due to general lack of knowledge about processes and traits underlying variation in competitive fertilization success. Here, we resolved processes determining sperm fate in twice‐mated females, using transgenic Drosophila simulans and Drosophila mauritiana populations with fluorescently labeled sperm heads. Comparisons among these two species and Drosophila melanogaster revealed a shared motif in the mechanisms of sperm precedence, with postcopulatory sexual selection potentially occurring during any of the three discrete stages: (1) insemination; (2) sperm storage; and (3) sperm use for fertilization, and involving four distinct phenomena: (1) sperm transfer; (2) sperm displacement; (3) sperm ejection; and (4) sperm selection for fertilizations. Yet, underlying the qualitative similarities were significant quantitative differences in nearly every relevant character and process. We evaluate these species differences in light of concurrent investigations of within‐population variation in competitive fertilization success and postmating/prezygotic reproductive isolation in hybrid matings between species to forge an understanding of the relationship between microevolutionary processes and macroevolutionary patterns as pertains to postcopulatory sexual selection in this group.